Proteins encoded at the left end of the adenovirus 2 (Ad2) genome in a transcription unit called E1A greatly stimulate transcription from at least seven of nine known Ad2 promoters for transcription by RNA polymerase II. Similar "transactivating" function have been described for viral proteins expressed immediately after infection in a number of DNA virus systems. E1A functions are also central to the process of oncogenic transformation by Ad2, and are representative of a class of oncogene products including polyoma and SV40 T- antigens, myc, N-myc and p53, all nuclear proteins which can cooperate with the c-Ha-ras 1 oncogene to completely transform primary embryo cells. The principal goals of the research proposed here are to understand the molecular mechanisms by which E1A proteins stimulate transcription from two Ad2 promoters, the E1B and IX promoters. The E1B promoter is active both before and after viral DNA replication in permissive cells and in transformed cells. Results of our unpublished studies on mutations constructed in the E1B promoter region suggest that the E1B promoter is unusually simple, being comprised of a single binding site for the Sp1 transcription factor (TF) and a TATA-box. No specific sequences appear to be required for E1A transactivation. Transcription from the IX promoter is only observed following viral DNA replication in permissive cells and is not observed in transformed cells. Yet the sequence of the IX promoter suggests that it too contains a single Sp1 site in close proximity to a TATA-box. This raises the question of what causes the difference in temporal regulation of these two Ad2 promoters. We propose to complete a mutational analysis of the E1B promoter and to perform a similar analysis of the IX promoter. We will assay the concentrations of TFs and other sequence specific DNA binding proteins which interact with the promoter elements identified by the mutational studies, their affinities for promoter sequences, and their specific activities for in vitro transcription in extracts from HeLa cells infected with Ad2 and E1A-mutants. Protein binding to these promoter elements in vivo will be assayed in cells infected with Ad2, E1A- mutants, and E1B and IX promoter mutants. Theses studies should provide some insight into the mechanism of transcription initiation at these promoters, its stimulation by E1A proteins, and temporal regulation of the IX promoter. A final specific aim is to design and construct temperature sensitive E1A proteins. Such ts E1A proteins would be useful tools in the study of transactivation and transformation by E1A proteins.